The broad, long-term objective of this proposal is to understand the pathogenesis of Lyme neuroborreliosis of the central nervous system (CNS). Inflammation in the CNS is thought to play a primary role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and the AIDS-dementia complex (ADC). Lyme borreliosis (LB), caused by the spirochete Borrelia burgdorferi, is primarily an inflammatory disease, and neuroborreliosis, i.e. LB of the nervous system, is a mild neurodegenerative disease. The central hypothesis to be explored in this grant proposal is that 1) B. burgdorferi spirochetes that have accessed the CNS can cause inflammation and 2) this inflammatory response may lead to neural injury, and ultimately loss of neural cells. Limited loss of these cells is viewed as a determinant of the neurologic impairment seen in Lyme neuroborreliosis. The secondary hypothesis to be examined is that spirochetal lipoproteins can induce, in part or in toto, the inflammatory effects of B. burgdorferi. More specifically, the cytokines IL-6, TNF-alpha, and IL-1beta, which have been implicated as possible effectors/mediators of neurodegeneration in AD and ADC, are elicited in the CNS by B. burgdorferi or its lipoproteins, and can mediate glial/neuronal loss. A corollary of our secondary hypotheses is that the innate immune response, in the form of a response to pathogen-associated molecular patterns (in this case lipoproteins) exerts via toll-like receptors (TLR) some or all of the inflammatory effects of B. burgdorferi. The following specific aims are proposed, using the rhesus macaque, the only animal model of LB of the CNS: SA-1: Experiments in vivo. SA-1a: assessment of inflammation and glial/neuronal loss in rhesus after intracerebral stereotaxic inoculations with live spirochetes and lipoproteins. Local cytokine secretion will be assessed by confocal microscopy using a novel procedure whereby cytokines are stained intracellularly with appropriate antibodies in freshly fixed brain slices treated ex vivo with Brefeldin A. The cell phenotype also will be identified, TLR expression determined, and glial/neuronal apoptosis verified in situ by the TUNEL assay. Spirochetal antigens will be localized with anti-lipoprotein antibodies. Neurons will be counted using image analysis software, and numbers compared with control animals; SA-1b: as with SA-1a, but following natural infections (by tick) of rhesus with B. burgdorferi; SA-1c: Archival fixed samples of Bb-infected rhesus brain and meninges will be assessed for inflammatory infiltrates and glial/neuronal apoptosis. Neurons will be counted differentially as above. SA-2: Experiments in vitro. SA-2a will assess the production of IL-1beta, TNF-alpha and IL-6 in single-cell-type and mixed primary cultures of rhesus glia stimulated with live B. burgdorferi and lipoprotein. The human neuroblastoma cell lines SK-N-SH and SH-SY5Y also will be employed, alone and in combination with rhesus glia, as well as rhesus neuronal-glial primary cultures; SA-2b will assess the role of these cytokines in eliciting glial/neuronal apoptosis; SA-2c will assess the expression and role of TLR in mediating cytokine production and apoptosis in the different cell combinations.